Hydrolases are well known as biocatalysts. In general, hydrolases can be used in the synthesis or transformation of acyl compounds like esters or amides. The products obtainable by the reactions find a broad spectrum of application. For example, hydrolases are well known for their use in the industrial hydrolysis or transesterification of triglycerides. Other examples are the production of pharmaceuticals or cosmetic ingredients, where stereoselectivity or high quality is of special interest. Production conditions for enzymatic reactions are generally milder and more specific than those of conventional chemical catalysis. Therefore, enzymatic reactions have received considerable and strongly increasing interest.
Industrially used hydrolases have been isolated from a broad variety of organisms, including bacteria, yeasts, higher animals and plants. However, most hydrolases have a limited operational temperature range, and are not suited for operations at increased temperatures.
There are several hydrolases, more specifically esterases, which can be used without deactivation at temperatures of about 60° C.; 70° C. being the absolute temperature maximum. An example is a lipase isolated from Candida antarctica which can be supplied in immobilised form. The absolute temperature maximum of this enzyme is 70° C. before the enzyme degradation becomes intolerably strong.
There are, however, numerous raw materials and products in the field of oleochemistry and surfactant chemistry which melt around, or well above, 65° C., e.g., stearic acid at 71° C. and behenic acid at 79° C. For carrying out enzymatic reactions without using solvents, which are generally undesired, and will also considerably increase the production costs, enzymes which are stable well above 60° C. are needed. Another advantage of performing enzymatic reactions at elevated temperatures is the viscosity reducing effect. This is especially useful if oligomers or polymers are converted by enzymatic reactions. In these cases, thermostable enzymes are prerequisites to conduct reactions at increased temperatures. A third aspect highlighting the advantages of thermostable enzymes, which is particularly important in the synthesis of surface active compounds, is reactions of hydrophilic and lipophilic compounds which need to be compatibilized to react with each other. For this purpose, a simple and efficient means is to increase temperature, which requires a suitable enzyme.
For an industrially useful hydrolase biocatalyst, excellent thermostability needs to be accompanied by broad substrate specificity and tolerance against different kinds of substrates and solvents. Indeed versatile catalysts are needed to fulfill the requirement of flexibility, which is essential for multipurpose production units often used in the production of specialty chemicals.
In the last two decades, the discovery and isolation of thermophilic microorganisms, such as eubacteria or archaea, isolated from, e.g., hot springs, or deep-sea hydrothermal vents, has resulted in the identification of new hydrolases which function at temperatures above 60° C. Hydrolases, especially esterases, can be characterized by different substrate specificities, substituent group or chain length preferences, and by unique inhibitors. See, for example, Barman, T. E. Enzyme Handbook, Springer-Verlag, Berlin-Heidelberg, 1969; Dixon, M. et al. Enzymes, Academic Press, New York, 1979. Hydrolases range from carboxylic ester hydrolases such as carboxyl esterases, lipases, phospholipases to peptide hydrolases and proteases. For example, lipases specifically act on long carbon chain substrates such as fats and oils, or generally speaking oleophilic substrates, whereas acylases are specific for short chain derivatives such as C2-C6 esters. Other very specific esterases are cholin esterases, steryl esterases, phospholipases A1 and A2 and many more. In many cases, these hydrolases are also known to show stereo- and regio-selective preferences resulting from the chiral nature inherent to protein active sites.
Hydrolases carry out their natural reactions, e.g., the hydrolysis of ester bonds in aqueous solutions. Under conditions that lack water, the reaction may be reversed and esters are formed from acids and alcohols. In vitro, these enzymes can be used to carry out reactions on a wide variety of substrates, including esters containing cyclic and acyclic alcohols, mono- and diesters, and lactams. See, for example, Santaniello, E., et al., “The biocatalytic approach leads to the preparation of enantiomerically pure chiral building blocks” (Chem. Rev. 92:1071-1140, 1992). By carrying out the reactions in the absence of water, the reactions of hydrolases can go in the reverse direction. The enzymes can catalyze esterification or acylation reactions to form ester or amide bonds (Santaniello, E. et al., supra). This process can also be used in the transesterification of esters, and in ring closure or opening reactions.
Hydrolases are a group of key enzymes in the metabolism of fats and are found in all organisms from microbes to mammals. In hydrolysis reactions, an ester or amide group is hydrolyzed to an organic acid and an alcohol or amine.
There are a number of industrial and scientific applications for hydrolases. In the following, some examples of the numerous uses for hydrolases are listed:    1) Hydrolases in the dairy industry as ripening starters for cheeses, such as the Swiss-type cheeses;    2) Hydrolases in the pulp and paper industry for lignin removal from cellulose pulps, for lignin solubilization by cleaving the ester linkages between aromatic acids and lignin and between lignin and hemicelluloses, and for disruption of cell wall structures when used in combination with xylanase and other xylan-degrading enzymes in biopulping and biobleaching of pulps;    3) Hydrolases in the synthesis of carbohydrate derivatives, such as sugar derivatives;    4) Hydrolases in combination with xylanases and cellulases, in the conversion of lignocellulosic wastes to fermentable sugars for producing a variety of chemicals and fuels;    5) Hydrolases as research reagents in studies on plant cell wall structure, particularly the nature of covalent bonds between lignin and carbohydrate polymers in the cell wall matrix;    6) Hydrolases as research reagents in studies on mechanisms related to disease resistance in plants and the process of organic matter decomposition;    7) Hydrolases in the selection of plant breeds for production of highly digestible animal feeds, particularly for ruminant animals;    8) Lipases in the hydrolysis of fats and oils to produce fatty acids; and    9) Lipases in the transesterification of fats and oils to produce special fats.
The term “hydrolase” as used in this application means that the enzyme belongs to the class E.C.3., according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), as found on www.chem.qmv.ac.uk/iubmb/enzyme/.
Most of the well-known hydrolases on the market, which are used as thermostable lipases or as thermostable hydrolases, are not adequate in stability against heat, and not practical for industrial applications at increased temperatures, broad pH-value ranges, or are not suited for long-term reactions. To date, only one hydrolase and a few lipases have been reported with moderately thermostable characteristics.
Ikeda, M. and D. S. Clark (Biotechnology and Bioengineering, 57, pp. 624-9, 1998) describe the molecular cloning of a thermostable hydrolase gene from the hyperthermophilic archaea Pyrococcus furiosus in E. coli. However, this document does not disclose the nucleic acid or amino acid sequences of this hydrolase, which functions only optimally in a narrow pH-value range of about 7.6, and shows good activity in a narrow tempera range only, with a temperature optimum at 100° C. Temperature stability could be shown with the following results for the half-life-time: 100° C. 34 h, 110° C. 6 h, 120° C. 2 h and 126° C. 50 min. The enzyme has only low or moderate lipase activity, and is more active to short hydrocarbon chain substrates. The expression of the plasmid containing the hydrolase DNA-sequence was very difficult due to low plasmid stability in liquid cultures of transformed E. coli. 
Tulin et al. (Biosci. Biotechn. Biochem., 57, pp. 856-857, 1993) reported a hydrolase derived from Bacillus stearothermophilus and cloned into Bacillus brevis. However, this hydrolase was only stable for up to 10 min at 70° C. Sugihara et al. (J. Biochem., 112, pp. 598-603, 1992) have isolated novel thermostable lipases from two microorganisms, a Bacillus soil isolate and a Pseudomonas cepacia soil isolate. The former lipase was stable up to 30 min at 65° C., but became rapidly inactivated above this temperature. The lipase from Pseudomonas cepacia was stable when heated for 30 min at 75° C. and pH 6.5, but had only 10% of its activity when assayed at this temperature.
Sigurgisladottir et al. (Biotechnol. Lett. 15, pp. 361-366, 1993) reported on the isolation of one Thermus stain and two Bacillus strains which contain lipases which act on olive oil at temperatures up to 80° C., although there was no report on enzyme stability in this study.
Hotta et al. (Appl. Environ. Microbiol. 68, pp. 3925-3931, 2002) found and characterised a thermostable hydrolase in the archaeon Pyrobaculum calidifontis. The hydrolase was shown to be thermostable for at least 2 h at 100° C. and to have a half-life-time at 110° C. of 56 in both measured in aqueous medium. The hydrolase is also fairly stable in the presence of water-miscible organic solvents. Its substrate specificity is limited to short hydrocarbon chain substrates, optimally for C6-chains.
The Japanese Unexamined Patent Publication No. Sho 62-79782 proposed a thermostable lipase. However, the optimum temperature of the enzyme is in the range of 60° C. to 70° C. and its thermostability is poor, since the residual activity after treatment at 70° C. for 15 min is below 10%.
European Patent Publication EP 0 117 553 A1 discloses a thermostable lipase derived from Rhizopus chinensis, but the optimum temperature of this enzyme is only 60° C. and the activity at a high temperature is fairly low.
Furthermore, there are reports on a lipase produced by Pseudomonas mephitica variety polytica which has an optimum temperature of 70° C. and is not inactivated by heat treatment at 60° C. for 14 h (Japanese Examined Patent Publication No. Sho. 50-25553), and a second lipase produced by Pseudomonas fraji which has an optimum temperature in the range of 75° C. to 80° C. and maintains 95% of the activity after heat treatment at 70° C. for 20 min (Agric. Biol. Chem. 41, 1353-1358, 1977). However, the temperature stability of these enzymes does not reach beyond 80° C. for 1 h, and therefore they are not sufficiently satisfactory with regard to thermostability.
U.S. Pat. No. 5,766,912 describes and claims a recombinant Humicola sp. lipase having a residual activity of at least 90% after 2 h at 60° C. and a pH from about 6 to about 9, and at least 80% after 2 h at 60° C. and at a pH from about 5.5 to about 9.2. This lipase has a residual activity of at least 95% after 2 h at 55° C. and at a pH from about 6 to about 9.5.
U.S. Pat. No. 5,173,417 describes a thermostable lipoprotein lipase from Streptomyces, which exhibits about 100% retention of the hydrolysing activity when treated in a buffer having a pH in the range of about 4 to 7 at about 60° C. for about 15 min, and a glycerol forming activity/fatty acid forming activity ratio of at least about 15%.
U.S. Pat. No. 5,306,636 describes a thermostable lipase from Pseudomonas sp KWI-56, which is stable for 24 h at pH 7.0 up to a temperature of 60° C. and which range of adaptable acting temperature with olive oil as the substrate is 60° C. to 65° C., with a pH optimum at 5.5-7.9. However, this lipase is deactivated to an extent of 82% after 24 h at 70° C.
U.S. Pat. No. 5,846,801 describes a thermostable lipase from Pseudomonas solanacearum having a temperature optimum of 80-90° C. (determined using triolein emulsion as a substrate), in a pH range of about 4-12, a pH optimum of 6.5-9.5. However, the residual activity of the enzyme had been measured within one hour only. Additionally, Pseudomonas solanacearum is a pathogenic microorganism.
U.S. Pat. No. 5,480,787 discloses a transesterification method using lipase powder. Preferably, a commercially available lipase from Alcaligenes is used for the transesterification of oils, fats and resins. However, no esterification or hydrolysis reactions are disclosed. Additionally, no disclosure with regard to amide formation by said enzyme is made. Further, the particle diameter of the immobilised hydrolase has to be controlled thoroughly during the enzymatic reactions, else the reactivity is reduced and the recovery of the lipase particles from the reaction liquid is difficult, which makes reuse impossible.
EP 0 709 465 A1 and EP 0 714 984 A1 describe a process for the production of optically active alcohols by a thermostable lipase derived from Alcaligenes under water-free conditions without a solvent. These publications, however, do not disclose the use of the lipase for hydrolysis or esterification reactions.
U.S. Pat. No. 5,714,373 discloses Thermococcus AV4 and enzymes produced by the same. This publication discloses the identification of a thermostable lipase, which is detected by the lipase activities, such as the hydrolysis of triglycerides to diglycerides and fatty acids, monoglycerides and glycerol. However, no examples for these reactions and no data on thermostability of the lipase are disclosed.
In the light of the prior art, it was desirable to provide hydrolases, and microorganisms producing the same, which combine several advantageous features that are not found in the enzymes of the prior art. Thus, it was necessary to provide a new type of hydrolases that have an excellent thermostability during long time reactions, with a high efficiency in a large pH range, in various media, and that are capable of acting on a broad range of substrates.